Among many environmental stress factors, heat stress can intensely deteriorate animal health, welfare, and productivity in poultry farming. In the present study, a novel cell culture comprised of different liver cell types from chickens was established to serve as a model for studying the effects of acute heat stress. Short, 1 h heat exposure strongly affected liver cells by increasing metabolism, triggering oxidative stress, and decreasing the generation of certain mediatory molecules of the cellular stress response. However, these alterations were normalized after 2 h of heat stress, suggesting a fast adaptation of liver cells. The results of this study underline the impact of short-term heat stress as a potential harmful factor affecting liver function in chickens.